The objective of this research is to understand the genetic and biochemical basis for maintaining the integrity of the mitochondrial compartment. The transfer of DNA from mitochondria to the nucleus is a fundamental evolutionary process for eucaryotic cells. Furthermore, the escape of DNA, RNA and protein from mitochondria significantly impacts the biology of cells and organisms with respect to development and disease. In order to define the genetic and biochemical basis for the escape of macromolecules from mitochondria, we have developed a unique and sensitive assay for identification and analysis of genes, designated YME, that affect the frequency of mitochondrial DNA (mtDNA) escape to the nucleus in the yeast Saccharomyces cerevisiae. YME2 encodes a mitochondrial membrane protein, Yme2p, that is of central importance to the process of mtDNA escape. When YME2 is mutated there are profound effects on mitochondrial function and cell viability. We have identified a genetic connection between Yme2p and proteins implicated in the chromosomal packaging of mtDNA. We hypothesize that Yme2p influences the ability of mtDNA to participate in many genetic processes and consequently is responsible for determining what fraction of mtDNA is able to escape from mitochondria. To test this hypothesis, we will investigate the effects of YME2 mutations on the physical and genetic status of mtDNA within mitochondria. The subcompartmental localization, relative concentration, and ability of mtDNA to participate in genetic processes such as recombination, mitotic and meiotic segregation and gene expression will be measured. Additionally, we will continue the genetic and biochemical analysis of YME2 to identify interacting genes and gene products. Extragenic and multicopy suppressors of yme2 growth and mtDNA escape phenotypes will be isolated. Complementary to this genetic approach, we will identify proteins that interact directly with Yme2p using the two-hybrid system co- immunoprecipitation, and co-purification of proteins that interact with Yme2p. Finally, we will isolate genes located downstream of YME2 in the mtDNA escape pathway. Several of these genes are identified by mutations that have interesting collateral phenotypes that will facilitate their isolation. The analysis of these genes will define the complex genetic and biochemical networks that maintain the compartmental and genomic integrity of mitochondria.